Nucleic acid sequences that can be used as primers and probes in the amplification and detection of all subtypes of HIV-1

ABSTRACT

The present invention is related to nucleic acid sequences that can be used in the field of virus diagnostics, more specifically the diagnosis of infections with the AIDS causing Human Immuno-deficiency Virus (HIV). With the present invention nucleotide sequences are provided that can be used as primers and probes in the amplification and detection of HIV-1 nucleic acid. The oligonucleotide sequences provided with the present invention are located in the LTR part of the HIV viral genome. It has been found that, by using the sequences of the present invention in methods for the amplification and detection of nucleic acid a sensitive and specific detection of HIV-1 can be obtained. The benefit of the sequences of the present invention primarily resides in the fact that, with the aid of primers and probes comprising the sequences according to the invention the nucleic acid of all presently known subtypes of HIV-1 can be detected with high accuracy and sensitivity. So far no primer pairs or hybridization probes have been developed that would allow the detection of such a broad range of HIV-1 variants. The oligonucleotide sequences according to the present invention are especially useful in methods for the amplification of nucleic acid.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/108,233, filed Apr. 18, 2005, which is a continuation of U.S.application Ser. No. 09/463,352, filed Jan. 21, 2000, now U.S. Pat. No.6,881,537, which is a 35 U.S.C. §371 national phase application ofInternational Application No. PCT/EP98/04945; filed on Aug. 5, 1998,which claims priority to European Patent Application No. 97202455.8,filed Aug. 8, 1997. The entire contents of each of these applications isfully incorporated herein by reference.

The present invention is related to nucleic acid sequences that can beused in the field of virus diagnostics, more specifically the diagnosisof infections with the AIDS causing Human immunodeficiency Virus (HIV).

Whereas conventional virus diagnosis has been based predominantly on thedetection of viral antigens or specific antibodies thereto, in recantyears attention has shifted towards methods for the direct detection ofthe genome of viruses or nucleic acid sequences derived thereof, bothRNA and DNA. These methods are usually based on nucleic acidhybridization. Nucleic acid hybridization is based on the ability of twostrands of nucleic acid containing complementary sequences to anneal toeach other under the appropriate conditions, thus forming a doublestranded structure. When the complementary strand is labeled, the labelcan be detected and is indicative for the presence of the targetsequence. Especially in combination with methods for the amplificationof nucleic acid sequences these methods have become an important tool inviral diagnosis, in particular for the detection of humanimmunodeficiency virus (HIV).

Nucleic acid amplification techniques are especially useful as anadditional technique in cases where serological methods give doubtfulresults or in cases where there may be a considerable time periodbetween infection and the development of antibodies to the virus. WithHIV, seroconversion usually can occur some 3-6 months after exposure tothe virus. Thus, whereas no antibodies will be detected withconventional immunoassays, proviral DNA or circulating viral RNA mayalready be detectable. Also in monitoring antiviral therapy, methodsbased on nucleic acid amplification have several advantages overserological methods. Especially quantitative amplification methodsprovide a powerful tool in assessing the changes in the amount of viruspresent before and during therapy.

The choice of the oligonucleotides to be used as primers and probes inthe amplification and detection of nucleic acid sequences is criticalfor the sensitivity and specificity of the assay. The sequence to beamplified is usually only present in a sample (for example a bloodsample obtained from a patient suspected of having a viral infection) inminute amounts. The primers should be sufficiently complementary to thetarget sequence to allow efficient amplification of the viral nucleicacid present in the sample. If the primers do not anneal property (dueto mispairing of the bases on the nucleotides in both strands) to thetarget sequence, amplification is seriously hampered. This will effectthe sensitivity of the assay and may result in false negative testresults. Due to the heterogeneity of viral genomes false negative testresults may be obtained if the primers and probes are capable ofrecognizing sequences present in only part of the variants of the virus.The HIV virus shows a high heterogeneity. Genetic variability has beendemonstrated amongst isolates from different continents but also betweenindividuals and between different stages of the disease. Based onsequence analysis two groups within HIV-1 have been identified: group M(M for “major”), and group O (O for “outlier”). Within group M subtypes(A-H), each constituting a phylogenetic separate set of sequences, havebeen assigned and additional ones are being Identified. This sequencevariation is not uniformly distributed throughout the genome. The HIV-1genome, like all retroviral genomes, roughly consists of the followingregions. The gag gene of the HIV-1 genome is the region encoding thecore proteins of the virus (for example, p24). The env gene encodes alarge precursor protein, gp160, which is processed into the envelopproteins gp120 and gp41. The pot gene encodes the polymerase of thevirus (reverse transcriptase). The Long Terminal Repeat region's (LTR's)are the regions on the viral genome that participate in the integrationof the virus with the host cell and in the regulation of transcriptionof the viral genes. Some regions are more prone to sequence variationthan others. Especially in the any domain sequence variation can be ashigh as 30% between members of the different subtypes. Ideally, primerselection should be based on knowledge of interstrain variability incandidate primer sequences and the consequences of mismatching at primersites. McCutchan et al, J. AIDS, 4, 1241-1250, 1991, used PCR to make agenetic comparison of different HIV-1 isolates. Using anchored PCR(varying sense primers were used with a constant antisense primer.primers were chosen from relatively conserved regions in gag, any andLTR) The effect of primer mispairing on the amount of PCR productobtained was also investigated. Mispairing at the 3′ end of the primerdecreased the amount of product sometimes more then 100-fold.

The detection of all presently known subtypes of HIV-1 is of extremeimportance, especially with regard to patient management, security ofblood and blood products and clinical- and epidemiological studies.Current assays for the amplification and subsequent detection of HIV-1derived nucleic acid sequences are usually based on amplification ofsequences in the gag region of the viral genome. These assays have beendeveloped for subtype B, which is the major subtype in Europeancountries and the United States. However, the presence of othersubtypes, which were geographically confined before, is increasing dueto frequent travel between these countries and, for example, Africancountries. Sensitive assays are therefore needed that are capable ofdetecting as much variants of the HIV-1 virus as possible (preferablyall).

Research aimed at identifying suitable primer sets for the reliableamplification of HIV-1 derived nucleic acid sequences has been ongoingfor the past years. Engelbrecht et al., J. Virol. Meth., 55, 391-400,1995, describe a study aimed at the development of a specific andsensitive PCR protocol using env, gag and LTR primer pairs to detectsubtypes present in the Western Cape, South Africa. Twenty four strainsof which it was known that they belonged to subtypes B, C and D wereanalyzed. It was found that the performance of the primer pairs wasgreatly dependent on the optimization of the reaction conditions for thedifferent primer pairs. Only when less stringent conditions were used(for example, with the LTR primer pair an increased cycle time and lowerannealing temperatures were required) these particular strains of HIVcould be detected with sufficient sensitivity and reproducibility withall primer pairs.

Zazzi et al., J. Med. Virol., 38, 172-174, 1992, developed a two-stepPCR reaction (using nested primers) for the detection of HIV-1 DNA inclinical samples. The primers used for amplification were derived fromthe gag gene and the LTR region. The patients tested in this study wereall from neighbouring areas., which makes it likely that they representonly a limited number of different viral strains.

A quantitative PCR method using LTR derived nested primers was describedby Vener et al. in BioTechniques, 21, 248-255, 1996. This procedure wasonly tested on HIV-1_(MN) Infected peripheral blood mononuclear cells(PBMC). Thus nothing can be said about the suitability of the primersused for detecting different subtypes of the virus.

With the present invention nucleotide sequences are provided that can beused as primers and probes in the amplification and detection of HIV-1nucleic acid. The oligonucleotide sequences provided with the presentinvention are located in the LTR part of the HIV viral genome. It hasbeen found that, by using the sequences of the present invention inmethods for the amplification and detection of nucleic acid a sensitiveand specific detection of HIV-1 can be obtained. The benefit of thesequences of the present invention primarily resides in the fact that,with the aid of primers and probes comprising the sequences according tothe invention the nucleic acid of all presently known subtypes of HIV-1can be detected with high accuracy and sensitivity. So far no primerpairs or hybridization probes have been developed that would allow thedetection of such a broad range of HIV-1 variants.

The oligonucleotide sequences according to the present invention areespecially useful in methods for the amplification of nucleic acid.

Various techniques for amplifying nucleic acid are known in the art. Oneexample of a technique for the amplification of a DNA target segment isthe so-called “polymerase chain reaction” (PCR). With the PCR techniquethe copy number of a particular target segment is increasedexponentially with a number of cycles. A pair of primers is used and ineach cycle a DNA primer is annealed to the 3′ side of each of the twostrands of the double stranded DNA-target sequence. The primers areextended with a DNA polymerase in the presence of the variousmononucleotides to generate double stranded DNA again. The strands ofthe double stranded DNA are separated from each other by thermaldenaturation and each strand serves as a template for primer annealingand subsequent elongation in a following cycle. The PCR method has beendescribed in Saiki et al., Science 230, 135, 1985 and in EuropeanPatents no. EP 200362 and EP 201184.

Another technique for the amplification of nucleic acid is the so-calledtranscription based amplification system (TAS). The TAS method isdescribed in International Patent Appl. no. WO 88/10315. Transcriptionbased amplification techniques usually comprise treating target nucleicacid with two oligonucleotides one of which comprises a promotersequence, to generate a template including a functional promoter.Multiple copies of RNA are transcribed from said template and can serveas a basis for further amplification.

An Isothermal continuous transcription based amplification method is theso-called NASBA process (“NASBA”) as described in European Patent no. EP329822. NASBA includes the use of T7 RNA polymerase to transcribemultiple copies of RNA from a template including a T7 promoter. Othertranscription based amplification techniques are described in EP 408295.EP 408295 is primarily concerned with a two-enzyme transcription basedamplification method. Transcription based amplification methods, such asthe NASBA method as described in EP 329822, are usually employed with aset of oligonucleotides, one of which is provided with a promotersequence that is recognized by a DNA dependent RNA polymerase such as,for example, T7 polymerase. Several modifications of transcription basedtechniques are known in the art. These modifications comprise, forexample, the use of blocked oligonucleotides (that may be provided witha promoter sequence). These olgo's are blocked so as to Inhibit anextension reaction proceeding therefrom (U.S. Pat. No. 5,554,516). Oneor more “promoter-primers” (oligonucleotides provided with a promotersequence) may be used in transcription based amplification techniques,optionally combined with the use of one or more oligonucleotides thatare not provided with a promoter sequence. For RNA amplification, atranscription based amplification technique, is a preferred technology.Amplification Using PCR, can also be based on an RNA template. Theactual PCR needs to be preceded by a reverse transcription step to copythe RNA into DNA (RT-PCR). However, if RT-PCR is used for the detectionof viral transcripts differentiation of mRNA- and DNA-derived PCRproducts is necessary. DNAse treatment prior to RT-PCR can be employed(Bitsch, A. et al., J. Infect. Dis 167, 740-743, 1993; Meyer, T. et al.,Mol. Cell. Probes. 8, 261-271, 1994), but sometimes fails to removecontaminating DNA sufficiently (Bitsch, A. et al., 1993).

In contrast to RT-PCR. NASBA, which is based on RNA transcription by T7RNA polymerase (Kievits et al., 1991; Compton, 1991), does not needdifferentiation between RNA- and DNA-derived amplification productssince it uses RNA as its principal target NASBA enables specificamplification of RNA targets even in a background of DNA.

The use of the oligonucleotides according to the invention is notlimited to any particular amplification technique or any particularmodification thereof. It is evident that the oligonucleotides accordingto the invention find their use in many different nucleic acidamplification techniques and various methods for detecting the presenceof (amplified) nucleic acid of HIV. The oligonucleotides of the presentinvention can likewise be used in quantitative amplification methods. Anexample if such quantitative method is described in EP 525882.

The term “oligonucleotide” as used herein refers to a molecule comprisedof two or more deoxyribonucleotides or ribonucleotides. Sucholigonucleotides may be used as primers and probes.

Of course, based on the sequences of the oligonucleotides of the presentinvention, analogues of oligonucleotides can also be prepared. Suchanalogues may constitute alternative structures such as “PNA” (moleculeswith a peptide-like backbone Instead of the phosphate sugar backbone ofnormal nucleic acid) or the like. It is evident that these alternativestructures, representing the sequences of the present invention arelikewise part of the present invention.

The term “primer” as used herein refers to an oligonucleotide eithernaturally occurring (e.g. as a restriction fragment) or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis of a primer extension product which is complementary to anucleic acid strand (template or target sequence) when placed undersuitable conditions (e.g. buffer, salt, temperature and pH) in thepresence of nucleotides and an agent for nucleic acid polymerization,such as DNA dependent or RNA dependent polymerase. A primer must besufficiently long to prime the synthesis of extension products in thepresence of an agent for polymerization. A typical primer contains atleast about 10 nucleotides in length of a sequence substantiallycomplementary or homologous to the target sequence, but somewhat longerprimers are preferred. Usually primers contain about 15-26 nucleotidesbut longer primers may also be employed, especially when the primerscontain additional sequences such as a promoter sequence for aparticular polymerase.

Normally a set of primers will consist of at least two primers, one‘upstream’ and one ‘downstream’ primer which together define theamplificate (the sequence that will be amplified using said primers).

Primarily for the use in transcription based amplification techniques,the oligonucleotides according to the invention may also be linked to apromoter sequence. The term “promoter sequence” defines a region of anucleic acid sequence that is specifically recognized by an RNApolymerase that binds to a recognized sequence and initiates the processof transcription by which an RNA transcript is produced. In principleany promoter sequence may be employed for which there is a known andavailable polymerase that is capable of recognizing the initiationsequence. Known and useful promoters are those that are recognized bycertain bacteriophage RNA polymerases such as bacteriophage T3, T7 orSP6. Olgonucleotides linked to a promoter sequence are commonly referredto as “promoter primers”. Their function as a primer, e.g. the startingpoint for an elongation reaction, however, may be blocked, as alreadymentioned above, or absent in some embodiments of transcription basedamplification reactions.

An oligonucleotide according to the present invention is substantiallycomplementary to a sequence of the LTR region of a nucleic acid sequenceof a HIV genome, said oligonucleotide being 10-50 nucleotides in lengthand comprising, at least a fragment of 10 nucleotides, of a sequenceselected from the group consisting of:

SEQ ID 1: G GGC GCC ACT GCT AGA GA SEQ ID 2: G TTC GGG CGC CAC TGC TAG ASEQ ID 3: CGGGCGCCACTGCTA SEQ ID 4: CTG CTT AAA GCC TCA ATA AA SEQ ID 5:CTC AAT AAA GCT TGC CTT GA SEQ ID 6: TCT GGT AAC TAG AGA TCC CTCSEQ ID 7: TAG TGT GTG CCC GTC TGT SEQ ID 8: AGT GTG TGC CCG TCT GTTSEQ ID 12: GAT GCA TGC TCA ATA AAG CTT GCC TTG AGTor the complementary sequence thereof.

It is understood that oligonucleotides consisting of the sequences ofthe present Invention may contain minor deletions, additions and/orsubstitutions of nucleic acid bases, to the extent that such alterationsdo not negatively affect the yield or product obtained to a significantdegree. Where oligonucleotides according to the present invention areused as probes, the alterations should not result in lowering thehybridization efficiency of the probe. For example, in case oftranscription based amplification techniques, wherein one or more of theprimers may be provided with a promoter sequence, the introduction of apurine-rich (=G or A) hybridizing sequence, Just after the promotersequence may have positive effects on the transcription (when there areC's and T's abortive transcription may occur). If no such sequence isavailable in the target nucleic acid a purine-rich sequence can beinserted in the oligonucleotide Just following the last three G residuesof the promoter sequence.

The sequences of the present invention are reflected as DNA sequences.The RNA equivalents of these sequences are likewise part of the presentinvention.

Preferred oligonucleotides according to the invention areoligonucleotides consisting essentially of a sequence selected from thegroup consisting of:

SEQ ID 1: G GGC GCC ACT GCT AGA GA SEQ ID 2: G TTC GGG CGC CAC TGC TAG ASEQ ID 3: CGGGCGCCACTGCTA SEQ ID 4: CTG CTT AAA GCC TCA ATA AA SEQ ID 5:CTC AAT AAA GCT TGC CTT GA SEQ ID 6: TCT GGT AAC TAG AGA TCC CTCSEQ ID 7: TAG TGT GTG CCC GTC TGT. SEQ ID 8: AGT GTG TGC CCG TCT GTT.SEQ ID 9:

G GGC GCC ACT GCT AGA GA SEQ ID 10:

G TTC GGG CGC CAC TGC TAG A SEQ ID 11:

CGGGCGCCACTGCTA SEQ ID 12: GAT GCA TGC TCA ATA AAG CTT GCC TTG AGTSEQ ID 9-11 actually comprise the sequence as reflected by SEQ ID 1-3.In SEQ ID 9-11, the sequences of SEQ ID 1-3 are operably linked to apromoter sequence (the T7 promoter sequence). This makes the sequencesespecially suitable for use as upstream primer in a transcription basedamplification technique such as NASBA.

A preferred embodiment of the present invention is a combination of twooligonucleotides according to the invention, for use as a set in nucleicacid amplification.

Such a pair of oligonucleotides, for use as a set in the amplificationof a target sequence located within the LTR region of the genome ofHIV-1, consists of a first oligonucleotide being 10-50 nucleotides inlength and comprising, at least a fragment of 10 nucleotides, of asequence selected from the group consisting of:

SEQ ID 1: G GGC GCC ACT GCT AGA GA SEQ ID 2: G TTC GGG CGC CAC TGC TAG ASEQ ID 3: CGGGCGCCACTGCTAand a second oligonucleotide being 10-50 nucleotides in length andcomprising, at least a fragment of 10 nucleotides, of a sequenceselected from the group consisting of:

SEQ ID 4: CTG CTT AAA GCC TCA ATA AA SEQ ID 5:CTC AAT AAA GCT TGC CTT GA. SEQ ID 12:GAT GCA TGC TCA ATA AAG CTT GCC TTG AGT

One of the oligonucleotides may serve as an “upstream oligonucleotide”,i.e., an upstream-primer, while the second oligonucleotide serves as a“downstream oligonucleotide”, i.e. downstream primer, in theamplification reaction. The location on the HIV-genome (or the sequencecomplementary thereto) to which both oligonucleotides comprised in sucha pair according to the Invention can anneal, will together define thesequence of the nucleic acid that is amplified. The amplified sequenceis located between the “primer-binding sites” within the LTR region ofthe HIV-genome. It has been found that, by using a pair ofoligonucleotides according to the invention in an amplificationreaction, accurate and reliable amplification of nucleic acid derivedfrom all presently know sub-types of HIV can be achieved.

A most preferred pair of oligonucleotides according to the inventionwill consist of a first primer comprising the sequence of SEQ ID NO 1and a second primer with the sequence of SEQ ID NO 5. For use in atranscription based amplification method, the oligonucleotide with SEQID NO 9 is preferred, in combination with an oligonucleotide with thesequence of SEQ ID NO 5.

Part of the oligonucleotides according to the invention are particularlysuitable for use as a probe in the detection of nucleic acid amplifiedwith a pair of oligonucleotides according to the invention. When used asa probe, said oligonucleotides may be provided with a detectable label.Olgonucleotides according to the invention that are especially suitableas a probe consist essentially of the sequence

SEQ ID 6: TCT GGT AAC TAG AGA TCC CTC SEQ ID 7: TAG TGT GTG CCC GTC TGTor SEQ ID 8: AGT GTG TGC CCG TCT GTTprovided with a detectable label. A most preferred oligonucleotide inthis respect is an oligonucleotide with a sequence as depicted in SEQ ID6.

Various labeling moieties are known in the art Said moiety may, forexample, either be a radioactive compound, a detectable enzyme (e.g.horse radish peroxidase (HRP)), a hapten like blotin, or any othermoiety capable of generating a detectable signal such as a colorimetric,fluorescent, chemiluminescent or electrochemiluminescent signal.

Hybrids between oligonucleotides according to the invention and(amplified) target nucleic acid may also be detected by other methodsknown to those skilled in the art.

Evidently methods for amplification of nucleic acid, like the ones thathave been mentioned above, using the oligonucleotides according to thepresent invention are also part of the invention.

The present invention further provides test kits for the amplificationand detection of HIV nucleic acid. The use of said test-kits enablesaccurate and sensitive screening of samples suspected of containing HIVderived nucleic acid. Such test-kits may contain a pair ofoligonucleotides according to the invention and optionally also aoligonucleotide according to the invention that can be used as a probefor the detection of the amplified material. Furthermore the test-kitmay contain suitable amplification reagents. These reagents are forexample the suitable enzymes for carrying out the amplificationreaction. A kit, adapted for use with NASBA, for example may containsuitable amounts of reverse transcriptase, RNase H and T7 RNApolymerase. Said enzymes may be present in the kit in a bufferedsolution but can likewise be provided as a lyophilized composition, forexample, a lyophilized spherical particle. Such lyophilized particleshave been disclosed in PCT appl. no. EP95/01288. The kit may further befurnished with buffer compositions, suitable for carrying out anamplification reaction. Said buffers may be optimized for the particularamplification technique for which the kit is intended as well as for usewith the particular oligonucleotides that are provided with the kit. Intranscription based amplification techniques, such as NASBA, saidbuffers may contain, for example, DMSO, which enhances the amplificationreaction (as is disclosed in PCT appl. no. US90/04733).

Furthermore the kit may be provided with an Internal control as a checkon the amplification procedure and to prevent the occurrence of falsenegative test results due to failures in the amplification procedure.The use of internal controls in transcription based amplificationtechniques is described in PCT appl. no. EP 93/102248. An optimalcontrol sequence is selected in such a way that it will not compete withthe target nucleic acid in the amplification reaction. Kits may alsocontain reagents for the isolation of nucleic acid from biologicalspecimen prior to amplification. A suitable method for the Isolation ofnucleic acid is disclosed in EP389063.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1:

Amplification products after blotting and detection by enhancedchemiluminescence obtained after amplification in presence (panel A) orabsence (panel B) of HIV-1 RNA using the following primer pairs. Lane 1:SEQ ID 9-SEQ ID 10, lane 2: SEQ ID 9-SEQ ID 5, lane 3: SEQ ID 10-SEQ ID4, lane 4: SEQ ID 10-SEQ ID 5, lane 5: SEQ ID 3-SEQ ID 12.

EXAMPLES Example 1 Amplification and Detection of HIV-1 Genomic RNA

The following procedures were applied to amplify and detect HIV-1genomic RNA from samples as described in the following examples.

Sample Preparation and Nucleic Acid Isolation.

The isolation of the nucleic acids was performed according to Boom etal., 1990, Journal of Clinical Microbiology 28, 495-503 and Europeanpatent no. EP0389083. In short: A volume of 200 μl of pooled plasma'sfrom healthy donors (negative for HBsAg, anti-HIV and anti-HCV) wasadded to 9001 of lysis buffer (47 mM Tris-HCl pH 7.2, 20 mM EDTA, 1.2%Triton X-100, 4.7 M guanidine thiocyanate (GuSCN, Fluka, Buchs,Switzerland)). After vortexing and centrifugation (30 seconds, 13,000rpm) a dilution series of HIV-1 RNA subtype B standard, characterisedand described by Layne et al., 1992, Virology 189, 695-714 or HIV-1positive specimens, was added. Upon addition of 50 μl activated silicon(0.4 g/ml suspension in 0.1 N HCl), the suspension was incubated for 10minutes at room temperature with regular vortexing. After centrifugation(30-60 seconds, 13,000 rpm) the silicon pellet was washed twice with 1ml of wash buffer (5.25 M GuSCN, 50 mM Tris-HCl pH 6.4), followed by two70% ethanol washes and one acetone wash step. Subsequently, the siliconpellets were dried during ten minutes in a 56° C. heating block. Thenucleic acids were eluted from the silicon by adding 50 μl elutionbuffer (1.0 mM Tris-HCl, pH 8.5) and incubation at 56° C. for 10minutes. Subsequently, 5 μl of the eluate was taken of the pellet forfurther use in the amplification reaction. The remaining eluate wasstored at −70° C.

NASBA Amplification.

Amplifications were carried out in a reaction volume of 20 μl which iscomposed of 10 μl primer mixture, 5 μl (isolated) nucleic acids and 5 μlenzyme mixture. The primer mixture was made by reconstitution of alyophilised accusphere into 50 μl accusphere diluent, 51.6 μl water, 8.4μl 2 M KCl and 5 μl of each primer (10 μM). The mixture was thoroughlyvortexed before use. From this primer mixture 10 μl was added to 5 μl ofthe (Isolated) HIV-1 RNA (standard). This mixture was Incubated for 5minutes at 65° C. and subsequently incubated at 41° C. for another 5minutes. After these incubations 5 μl enzyme mixture was added andincubated for 5 minutes at 41° C. The tubes were transferred to thedetection area and incubated for 90 minutes at 41° C. After theamplification reaction the tubes were stored at −20° C. until furtheruse.

Each amplification reaction contained the following reagents:

40 mM Tris-HCl, pH 8.5

12 mM MgCl₂

70 mM KCl

15% v/v dimethyl sulfoxide

5 mM dithiothreitol

1 mM of each deoxynucleoside triphosphate

2 mM of the nucleosides rATP, rCTP, rUTP

1.5 mM rGTP

0.5 mM ITP

0.105 μg/μl BSA

0.08 units RNaseH

32 units T₇ RNA polymerase

6.4 units avian myloablastosis virus reverse transcriptase (Seikagaku,USA)

0.2 μM of each primer

375 mM sorbitol

45.6 mM sucrose

28.5 mM mannitol

0.13 mM dextran T40

Detection of the Amplified Products.

A. Gel Electrophoresis.

The presence of amplified products was analysed using an agarose gel(100 ml of 2% Pronarose and 0.5 μg/ml ethidium bromide) and 1*TAE (40 mMTris-acetate, 1 mM EDTA pH 8.0) running buffer are used. Electrophoresiswas carried out at 100 volts for approximately 30 minutes. The ethidiumbromide-stained bands of the amplified products were visualised using UVradiation. The blot was hybridised with biotin probe (3 μM) inhybridisation mix (750 mM NaCl, 75 mM sodumnitrate, 20 mM NatHPO₄NaH₂PO₄(pH 6.7), 10*Denhardts) by Incubating the blot for 4 hours at 50° C.After hybridisation the blot was washed two times for 5 minutes at 50°C. with 450 mM NaCl, 45 mM sodiumcitrate pH 6.4 (2*SSPE) and 1% sodiumdodecyl sulphate (SDS) solution and one time for 10 minutes with 20 mMNaeHPO₄ pH 7.4, 360 mM NaCl, 2 mM EDTA and 0.1% SDS at room temperature.Subsequently, the blot was incubated for 30 minutes with 2 μlstreptavidin/horse radish peroxidase solution (500 U/ml from theenhanced chemiluminiscense detection kit of Amersham Life Science) in 10ml 50 mM Na₂HPO₄. 900 mM NaCl, 5 mM EDTA, pH7.4 and 0.5% SDS. Followingwashes respectively two times for 5 minutes in 2*SSPE, 0.1% SDS and oncefor 10 minutes in 2*SSPE at room temperature the blot was dried betweentissues, developed and exposed to a film according to the Amersham kitprotocol.

B. Electrochemiluminescence (ECL) Probe Hybridisation.

The amplification products were diluted two times in detection diluent(1.0 mM Tris/HCl, pH 8.5 and 0.2 g/l 2-methyllsothiazolone HCl).Subsequently, 5 μl of the diluted amplification product was incubatedfor 30 minutes at 41° C. with 0.084 μM of the HIV-1 specific blotinprobe bound to 5 μg streptavidin-coated magnetic beads (mean size 2.8μm±0.2 μm, Dynal, Great Neck, N.Y., USA) and 2*10¹¹ molecules of an ECL(Tris(2,2-bipyridine) ruthenium [II] complex)-labelled probe, in a totalvolume of 25 μl 750 mM NaCl, 75 mM sodumcirate, pH 6.4 (5*SSC). Asnegative controls, detection diluent was also incubated with thebead-probe and ECL-probe mixtures. During incubation, tubes wereagitated every 10 minutes to keep the beads in suspension. Subsequently,300 μl of assay buffer solution (100 mM tripropylamine, pH 7.5) wasadded and the tubes were placed in an ECL detection instrument (NASBAQR-system from Organon Teknika BV) for reading the emitted ECL signals.

Example 2 Amplification and Detection of HIV-1 Genomic RNA

The following primer pairs were tested on 10⁴ copies (as determined byspectrophotometry at 260 nm) of the HIV-1 RNA standard. The RNA wasadded directly into the amplification. The analysis of the amplifiedproducts was performed by gel electophoresis as described in example 1.The results are shown in the FIG. 1.

All primer pairs and probes of the present invention were able toamplify and detect HIV-1 RNA from subtype B to a similar extentAnalytical sensitivity is shown for two primer pairs (SEQ ID 9/SEQ ID 5and SEQ ID11/SEQ ID 5) using a dilution series of the HIV-1 RNA standardwhich has an initial concentration of 5.5*10⁹ copies/ml. The detectionwas done with probes having the sequences as depicted in SEQ ID 7 andSEQ ID 6. The amplification and detection was carried out as describedin example 1. Table 1 shows the results obtained with both primer pairs.

TABLE 1 HIV-1 RNA SEQ ID 3-SEQ ID 5 SEQ ID 9-SEQ ID 5 copies No.detected % detected No. detected % detected 200 4:4 100% 4:4 100% 1008:8 100% 8:8 100% 50 8:8 100% 8:8 100% 25 7:8 87.5%  7:8 87.5%  12 6:8 75% 4:8  50% 6 2:8  25% 4:7  57% 3 0:8  0% 2:7 28.5%  0 0:8  0% 0:7  0%

Both primer pairs have approximately the same sensitivity for the HIV-1RNA standard respectively a detection rate of 70% for the primer pairSEQ ID 9/SEQ ID 5 and a detection rate of 63% for the primer pair SEQ ID3/SEQ ID 5.

Example 3 Amplification and Detection of HIV-1 RNA in Presence of anInternal Control

In this example the primer pair SEQ ID 9/SEQ ID 5 and probe with thesequence of SEQ ID 7 was tested on dilution series of the HIV-1 RNAstandard in presence of an Internal control (ic-) RNA. The ic-RNA is anin vitro transcript which contains part of the LTR sequence of HIV-1HXB-2 and 10⁴ copies (as determined by spectrophotometry at 260 nm) areadded prior to nucleic acids isolation. The isolation, amplification andECL detection was performed as described in example 1. As a comparison,separate amplification and detection was performed using a primer pairlocated in the HIV-1 gag region (P1/P2) previously described by VanGemen et al, 1993, Journal of Virological Methods 43, 177-188. Theprobes used to detect the amplification products generated by the gagbased primer pair were:

Biotin probe: 5′-TGTTAAAAGAGACCATCAATGAGGA. ECL probe:5′-GAATGGGATAGAGTGCATCCAGTG.

Results are shown in Table 2.

TABLE 2 HIV-1 RNA copies/ LTR SEQ ID 9-SEQ ID 5 GAG P1/P2 200 μl No.detected % detected No. detected % detected 320 9:10 90% nt* nt* 1605:10 50% 8:10 80% 80 5:10 50% 5:10 50% 40 3:8  38% 4:10 40% 20 3:10 32%1:10 10% 10 1:10 10% 2:10 20% 0 0:5   0% 1:5  20% *nt is not tested.

Both the LTR primer pair of this example and the gag primer pair wereable to detect a similar amount of HIV-1 RNA in an assay controlled byan in vitro produced RNA which was added prior to the nucleic acidsisolation.

Example 4 Detection of HIV-1 RNA in HIV-1 Positive Samples

In this example, 40 HIV-1 positive samples from various geographicallocations were analysed for presence of HIV-1 RNA. All samples wereisolated by the nucleic acids isolation method described in example 1.Amplification and detection were carried out as described in example 1using primer pair SEQ ID 9/SEQ ID 5 and probe with the sequence of SEQID 7 or probe with the sequence of SEQ ID 8.

For comparison, separate amplification and detection was carried outusing the gag primer pair and probes as described in example 3. Presenceof amplified products was detected by ECL probe hybridisation accordingto the method of example 1. Result are shown in Table 3.

TABLE 3 Primer pair ECL Probe No. Detected % Detected. SEQ ID 9/SEQ ID 5SEQ ID 7 40/40 100% (LTR) SEQ ID 9/SEQ ID 5 SEQ ID 8 10/10 100% (LTR)P1/P2 (gag) 29/40 72.5% 

Both primer probe combinations derived from the LTR region of HIV-1described in this example were able to detect HIV-1 from all the samplestested. In contrast, the primer probe combination of the gag assayfailed to detect HIV-1 genomic RNA from a great number of samples.

Example 5 Amplification and Detection of HIV-1 RNA with Defined Subtypes

In this example, 33 samples harbouring HIV-1 RNA of known envelope-basedsubtypes were tested. The samples originate from subtyped virusespropagated in cell cultures. Samples of cell culture supernatants fromboth variants of group M subtypes A through H and variants of group Owere spiked to HIV-1 negative human plasma and treated according to themethod from example 1. Amplification and detection was performedessentially as example 4. Results are shown table 4 as the number ofsamples of each type from which HIV-1 RNA was detected out of the totalnumber of each type tested.

TABLE 4 HIV-1 Types Primer pair A B C D E F G H O Total SEQ ID 9/SEQ ID5 5:5 2:2 2:2 5:5 7:7 1:1 1:1 1:1 9:9 33:33 (LTR) 4:5 2:2 2:2 5:5 6:71:1 1:1 1:1 0:9 22:33

HIV-1 RNA was detected from all 33 HIV-1 samples using LTR primer pairSEQ ID 9/SEQ ID 5 and probe with sequence of SEQ ID 7. In contrast, theassay using the gag primers probes combination as described in example 3failed to detect subtype A and subtype E each from one of the samplesand all samples containing HIV-1 RNA from group O members.

Example 6 Amplification and Detection of HIV-1 Clinical Samples

In this example, 7 samples obtained from seropositive patientsoriginating from Africa, South America and Asia were assayed for thepresence of HIV-1 genomic RNA. Despite the fact that blood samples fromthese patients are both anti-p24 antibody (Abbott Laboratories, AbbottPark, Ill.) and western blot (Genelabs) positive, all specimens werescored negative by the NASBA HIV-1 RNA QL assay (Organon Teknika BV)which uses the gag primer pair and probe combination from example 3 andonly a single sample (R9612222) was detected and quantitated (29.500 RNAcopies/ml) by the Quantiplex HIV-1 RNA 2.0 (Chiron) assay using a samplevolume of 50 μl. In contrast, using an equal sample volume the primerpair SEQ ID 9/SEQ ID 5 and the probe with sequence of SEQ ID 7 detectedfour out of seven samples (Table 5).

TABLE 5 SEQ ID 9/SEQ ID 5 Sample code Country (LTR) R9610155 Thailandpositive R9612222 Ghana positive R9700062 Brazil negative R9612218 Zairenegative R9611710 Liberia positive R9610718 Antilles positive R9607884Rwanda negative

The samples missed by the LTR primer probe combination are not detecteddue to a low viral load below the detection level since by using asample volume of one ml the Quantiplex HIV-1 RNA 2.0 (Chiron) assayquantitated HIV-1 RNA levels of 30 or below 30 HIV-1 RNA copies per 50μl of these samples.

The invention claimed is:
 1. A method for amplifying HIV-1 nucleic acidin a sample, comprising: a) contacting the sample with a pair ofoligonucleotide primers that bind to a first primer binding site and asecond primer binding site located within the LTR region of the HIV-1genome; and b) performing a nucleic acid amplification under conditionswherein said oligonucleotide primers bind only to said first and secondprimer binding sites, thereby amplifying HIV-1 nucleic acid in thesample; wherein said pair of oligonucleotide primers consists of a firstprimer and a second primer, wherein said first primer consistsessentially of a first oligonucleotide that is fully complementary to asequence of the LTR region at the first primer binding site, saidoligonucleotide being 15-26 nucleotides in length and comprising atleast 15 sequential nucleotides of the nucleotide sequence of:SEQ ID NO: 1:  G GGC GCC ACT GCT AGA GA;

said first oligonucleotide being operably linked to a promoter; andwherein said second primer consists essentially of a secondoligonucleotide that is fully complementary to a sequence which is thereverse complement of a sequence of the LTR region at the second primerbinding site, said oligonucleotide being 10-26 nucleotides in length andcomprising at least 10 sequential nucleotides of the nucleotide sequenceof: SEQ ID NO: 5:  CTC AAT AAA GCT TGC CTT GA.


2. The method of claim 1, wherein said second oligonucleotide that isfully complementary to a sequence which is the reverse complement of asequence of the LTR region is 15-26 nucleotides in length and comprisesat least 15 sequential nucleotides of the nucleotide sequence of:SEQ ID NO: 5:  CTC AAT AAA GCT TGC CTT GA.


3. The method of claim 1, wherein said first oligonucleotide comprisesthe nucleotide sequence of: SEQ ID NO: 1:  G GGC GCC ACT GCT AGA GA.


4. The method of claim 1, wherein performing a nucleic acidamplification comprises contacting the sample with one or more probeoligonucleotides selected from: SEQ ID NO: 6: TCT GGT AAC TAG AGA TCC CTC SEQ ID NO: 7:  TAG TGT GTG CCC GTC TGTSEQ ID NO: 8:  AGT GTG TGC CCG TCT GTT

one or more of which are provided with a detectable label, underconditions where hybridization can occur; and detecting the presence ofthe label in any hybrids formed between the amplified sequence and theone or more probe oligonucleotides.
 5. The method of claim 1, whereinsaid promoter is a T3, T7, or SP6 promoter.
 6. A method for amplifyingHIV-1 nucleic acid in a sample, comprising: a) contacting the samplewith a primer set that bind to a first primer binding site and a secondprimer binding site located within the LTR region of the HIV-1 genome;and b) performing a nucleic acid amplification under conditions whereinsaid oligonucleotide primers bind only to said first and second primerbinding, thereby amplifying HIV-1 nucleic acid in the sample; whereinsaid primer set consists of a first primer and a second primer, whereinsaid first primer consists essentially of a first hybridizingoligonucleotide that is fully complementary to a sequence of the LTRregion at the first primer binding site, said oligonucleotide being15-26 nucleotides in length and comprising at least 15 sequentialnucleotides of the nucleotide sequence of: SEQ ID NO: 1: G GGC GCC ACT GCT AGA GA;

said first hybridizing oligonucleotide being operably linked to apromoter; and wherein said second primer consists essentially of asecond hybridizing oligonucleotide that is fully complementary to asequence which is the reverse complement of a sequence of the LTR regionat the second primer binding site, said oligonucleotide being 10-26nucleotides in length and comprising at least 10 sequential nucleotidesof the nucleotide sequence of: SEQ ID NO: 5: CTC AAT AAA GCT TGC CTT GA.


7. The method of claim 6, wherein said second oligonucleotide that isfully complementary to a sequence which is the reverse complement of asequence of the LTR region is 15-26 nucleotides in length and comprisesat least 15 sequential nucleotides of the nucleotide sequence of:SEQ ID NO: 5:  CTC AAT AAA GCT TGC CTT GA.


8. The method of claim 6, wherein said first oligonucleotide comprisesthe nucleotide sequence of: SEQ ID NO: 1:  G GGC GCC ACT GCT AGA GA.


9. The method of claim 6, wherein performing a nucleic acidamplification comprises contacting the sample with one or more probeoligonucleotides selected from: SEQ ID NO: 6: TCT GGT AAC TAG AGA TCC CTC SEQ ID NO: 7: TAG TGT GTG CCC GTC TGTSEQ ID NO: 8:  AGT GTG TGC CCG TCT GTT

one or more of which are provided with a detectable label, underconditions where hybridization can occur; and detecting the presence ofthe label in any hybrids formed between the amplified sequence and theone or more probe oligonucleotides.
 10. The method of claim 6, whereinsaid promoter is a T3, T7, or SP6 promoter.
 11. A method for amplifyingHIV-1 nucleic acid in a sample, comprising: a) contacting the samplewith a pair of oligonucleotide primers that bind to a first primerbinding site and a second primer binding site located within the LTRregion of the HIV-1 genome; and b) performing a nucleic acidamplification under conditions wherein said oligonucleotide primers bindonly to said first and second primer binding sites, thereby amplifyingHIV-1 nucleic acid in the sample; wherein said pair of oligonucleotideprimers consists of a first primer and a second primer, wherein saidfirst primer consists essentially of a first hybridizing oligonucleotidethat is fully complementary to a sequence of the LTR region at the firstprimer binding site, said oligonucleotide being 15-26 nucleotides inlength, wherein said first hybridizing oligonucleotide comprises atleast 15 sequential nucleotides of the nucleotide sequence of:SEQ ID NO: 1:  G GGC GCC ACT GCT AGA GA;

said hybridizing oligonucleotide being operably linked to a promoter;and wherein said second primer consists essentially of a secondhybridizing oligonucleotide that is fully complementary to a sequencewhich is the reverse complement of a sequence of the LTR region at thesecond primer binding site, said oligonucleotide being 10-26 nucleotidesin length, wherein said second hybridizing oligonucleotide comprises atleast 10sequential nucleotides of the nucleotide sequence of:SEQ ID NO: 5:  CTC AAT AAA GCT TGC CTT GA.


12. The method of claim 11, wherein said second oligonucleotide that isfully complementary to a sequence which is the reverse complement of asequence of the LTR region is 15-26 nucleotides in length and comprisesat least 15 sequential nucleotides of the nucleotide sequence of:SEQ ID NO: 5:  CTC AAT AAA GCT TGC CTT GA.


13. The method of claim 11, wherein said first oligonucleotide comprisesthe nucleotide sequence of: SEQ ID NO: 1:  G GGC GCC ACT GCT AGA GA.


14. The method of claim 11, wherein performing a nucleic acidamplification comprises contacting the sample with one or more probeoligonucleotides selected from: SEQ ID NO: 6: TCT GGT AAC TAG AGA TCC CTC SEQ ID NO: 7:  TAG TGT GTG CCC GTC TGTSEQ ID NO: 8:  AGT GTG TGC CCG TCT GTT

one or more of which are provided with a detectable label, underconditions where hybridization can occur; and detecting the presence ofthe label in any hybrids formed between the amplified sequence and theone or more probe oligonucleotides.
 15. The method of claim 11, whereinsaid promoter is a T3, T7, or SP6 promoter.